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Summary

Cultures of the non-heterocystous cyanobacterium, Leptolyngbya nodulosa, could be grown indefinitely in media devoid of combined nitrogen. Acetylene reduction assays showed that these cultures fixed nitrogen in the dark period of a diurnal cycle under micro-oxygenic or anaerobic conditions. Addition of DCMU to cultures induced much higher rates of nitrogenase activity, most of which occurred in the light. Measurements of activity in the presence of chloramphenicol indicated that nitrogenase is synthesized in darkness and probably destroyed in the subsequent light period. Neither the dark-mediated nitrogenase in the absence of DCMU nor light-mediated activity in the presence of DCMU could be sustained for more than 3 days without a photoperiodic light/dark cycle. Axenic cultures could not be grown in the absence of combined nitrogen and did not demonstrate any acetylene reduction activity. An identical nifH gene sequence was found in axenic and non-axenic cultures of L. nodulosa. RT-PCR demonstrated that this gene was expressed only in non-axenic cultures. Western blotting showed that the Fe-protein of nitrogenase is absent in cultures that are incapable of acetylene reduction, indicating that the lack of nitrogenase activity is likely due to the absence of the enzyme. These observations strongly indicate that L. nodulosa contains a functional nitrogenase which is not expressed in the absence of heterotrophic bacteria.

Supporting Information

Fig. S2. Neighbour-Joining tree of a partial nifH sequence was generated with Phylip. Rhizobium nifH was used as the outgroup. A global blast of the NCBI GenBank database (15 September 2009) showed that the closest matches to the nifH sequence in Leptolyngbya nodulosa are putative nifH genes from marine ‘uncultured bacteria’. No published information indicated whether the uncultured bacteria were cyanobacteria or heterotrophic bacteria. The most similar authentic cyanobacterial match is to Leptolyngbya sp. PCC 7104. Numbers after strain names are GenBank accession numbers.

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Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.